Pharmaceutical composition for prophylaxis and therapy of diseases associated with ocular fundus tissue cytography

ABSTRACT

A pharmaceutical composition for the prophylaxis and therapy of a disease arising from ocular fundus tissue cytopathy such as retinochoroidal disease, glaucoma, and posterior complication arising form photocoagulation, which contains, as an active ingredient, a compound of formula (I) wherein R 1  represents an alkyl group having 1 to 4 carbon atoms or an aryl group having 6 to 10 carbon atoms which is optionally substituted; R 2  and R 3  may be the same or different and each represents hydrogen or an alkyl group having 1 to 4 carbon atoms or R 2  and R 3  may jointly form a ring having 3 to 7 carbon atoms; and R 4  represents a lower alkyl group which substituted by aryl, cycloalkyl, or aromatic heterocyclic residue, or a pharmaceutically acceptable salt thereof.

This is a 371 application of PCT/JP99/01031 filed Mar. 3, 1999.

TECHNICAL FIELD

The present invention relates to a drug for the prophylaxis and therapyof the diseases associated with ocular fundus tissue cytopathy.

BACKGROUND ART

The ocular fundus tissue comprises the retina, retinal vessels, retinalpigment epithelium, optic disc, choroid, and sclera and constitutes anorgan for transmitting light stimuli from the crystalline lens to thebrain through optic nerve cells. Therefore, injury of the ocular fundustissue cells induces serious diseases leading to blindness in manycases. Impairment of ocular fundus tissue cells is frequently caused byischemia and changes of various biological substances, such as elevationof excitatory amino acid levels and decreases in ATP. Moreover, theocular fundus tissue cells are damaged by systemic diseases such ashypertension and diabetes, aging, and trauma as well. While laserphotocoagulation is used for inhibition or elimination of neogeneticvessels in diabetic retinopathy and macular degeneration, elimination ofvessel occlusion in retinal vessel occlusion, and the like, ocularfundus tissue cytopathy caused by changes in the circulation in thevicinity of the laser irradiation site, inflammatory reaction due to theheat of photocoagulation and the like poses problems.

The diseases associated with ocular fundus tissue injuries develop dueto the various factors mentioned above or a complicated combination ofvarious factors. The disease arising from injuries to the ocular fundustissue includes retinochoroidal disease (e.g. retinal vascularabnormalities such as retinal vessel occlusion, retinal periphlebitis,Eales' disease, ischemic eye syndrome, retinal arteriolar macroaneurysm,etc.; retinopathy associated with hypertension or renal disease;diabetic retinopathy; retinal pigment epithelitis; retinal dystrophy;macular dystrophy; retinochoroidal atrophy; retinochoroiditis; maculardegeneration; macular edema; retinal pigment epithelial detachment;retinal detachment; degenerative retinoschisis; tumors such asretinoblastoma, tumors of the retinal pigment epithelium, capillaryhemangioma of the optic nerve head, etc.; optic neuropathies such asischemic optic neuropathy etc.; optic disc swelling such as chokeddisc/papilledema etc.); and glaucoma associated with ocular fundustissue cytopathy (e.g. open angle glaucoma, low tension glaucoma, angleclosure glaucoma, etc.); and further the posterior complications arisingfrom photocoagulation such as macular edema, retinal detachment, opticneuritis, abnormal visual field, abnormal light sense, and color visiondefect. As used herein, by the posterior complications arising fromphotocoagulation is meant ocular fundus tissue cytopathy, caused bychanges in the circulation in the vicinity of laser irradiation site,which is attributable to photocoagulation upon laser irradiation,inflammatory reaction due to the heat of photocoagulation, and diseasesinduced by these disorders.

As the current drug therapy of diseases associated with ocular fundustissue cytopathy, microcirculation improving agents such as tocopherolnicotinate, which is a vitamin E preparation, pentoxifylline, etc.,various steroidal drugs, antiprostaglandins, and antiphologistic enzymepreparations are administered orally. However, those therapies areeither not effective enough or have side-effect problems such ashypotension and gastrointestinal disorders. As therapeutic modalitiesfor glaucoma, cholinergic agonists represented by pilocarpine,sympathomimetic drugs such as epinephrine, dipivefrin, etc., andβ-adrenergic antagonists such as timolol, pindolol, carteolol, etc. areavailable for topical administration (e.g. eye-drops) but various sideeffects associated with their mechanisms of action are problems.

Recently, it has been reported that compounds having calpain inhibitoryactivity have an action to inhibit ischemic cell death [Lee K., FrankS., Vanderklish, P., Arai A., Lynch G., Proc. Natl. Acad. Sci., 88,7233-7237 (1991), Rami A., Krieglstein J., Brain Res., 609, 67-70(1993)]. However, those reports describe only the inhibitory effect oncell death in connection with the death of neurons in the hypocampus anddo not indicate the inhibitory effect of the drugs on ocular fundustissue cytopathy or their utility in the field of ophthalmology.Moreover, the inhibitors so far used have many shortcomings in theaspect of transfer to tissues and adverse reactions. EP0771565 statesthat a cysteine protease inhibitor is useful for neovascularization indiabetic retinopathy etc. but does not even hint at diseases associatedwith ocular fundus tissue cytopathy.

The present invention has for its object provision of a drug for theprophylaxis and therapy of ocular fundus tissue cytopathy, whichovercomes the above-mentioned disadvantages. It is a further object ofthe present invention to provide a drug for the prophylaxis and therapyof diseases arising from ocular fundus tissue cytopathy and a method forthe prevention and treatment of such diseases.

It should be understood that, in the context of the present invention,the term “ocular fundus tissue” means to include the retina, retinalblood vessels, retinal pigment epithelium, optic disc, choroid, sclera,and vitreous base, and that, of these tissues, the retina, retinal bloodvessels, retinal pigment epithelium, optic disc, and choroid aresometimes referred to collectively as the retinochoroid.

DISCLOSURE OF THE INVENTION

The inventors of the present invention endeavored to develop a drug forthe prophylaxis and therapy of ocular fundus tissue cytopathy, which iseffective for improving ocular fundus tissue cytopathy and which issafe, and found that a compound of the following formula (I)

wherein R¹ represents an alkyl group having 1-4 carbon atoms or an arylgroup having 6-10 carbon atoms which is optionally substituted; R² andR³ may be the same or different and each represents hydrogen or an alkylgroup having 1-4 carbon atoms or R² and R³ may jointly form a ringhaving 3-7 carbon atoms; R⁴ represents a lower alkyl group which isoptionally substituted by aryl, cycloalkyl, or aromatic heterocyclicresidue, and a pharmaceutically acceptable salt thereof exhibitremarkable prophylactic or therapeutic efficacy against ocular fundustissue cytopathy.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagrammatic representation of the effect on injury of ratretinal ganglion cells. The ordinate represents the number of ganglioncells. In the diagram, a) denotes a significant difference (p<0.01) fromthe normal group by Student's t-test; b) denotes a significantdifference (p<0.01) from the control group by Student's t-test.

FIG. 2 is a diagrammatic representation of the effect on injury of therat retinal inner nuclear layer cells. The ordinate represents thenumber of inner nuclear layer cells. In the diagram, a) denotes asignificant difference (p<0.01) from the normal group by Student'st-test; b) denotes a significant difference (p<0.01) from the controlgroup by Student's t-test.

FIG. 3(a) and FIG. 3(b) are SDS electrophoretic images showing thecleavage product of endocellular spectrin obtained upon homogenation ofretina tissues detected after incubation of rat retina tissues underhypoxia, wherein FIG. 3(a) and FIG. 3(b) show SDS electrophoretic imagesafter incubation for 3 hours and 6 hours, respectively. In the Figures,M is a molecular weight marker, 1 shows retina tissues incubated in aglucose-containing culture medium in the presence of oxygen, 2 showsretina tissues incubated under hypoxia in a culture medium withoutglucose, 3 shows retina tissues incubated under hypoxia in a culturemedium without glucose but supplemented with 100 μM ofN-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (hereinafter abbreviatedas Compound 1), and 4 shows retina tissues incubated under hypoxia in aculture medium without glucose but supplemented with 100 μM ofCbz-Val-Phe-H [hereinafter abbreviated as MDL]. Protein band of 220 kDashows intact molecule of spectrin, and 145 and 150 kDa bands show thecleavage products of spectrin.

The compound of formula (I) for use in this invention and apharmaceutically acceptable salt thereof are known compounds disclosedin Japanese Patent Unexamined Publication No. 43464/1997 (EP0771565) andcan be produced typically by the processes described therein.

Referring to formula (I), when the amino acid moieties exist as opticalisomers, they are L-isomers unless otherwise indicated.

Referring further to formula (I), the C₁-C₄ alkyl group for R¹ includesmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyland the like. Of these, methyl is preferred.

The C₆-C₁₀ aryl group for R¹ includes phenyl, naphthyl, indenyl,azulenyl, and so forth. Preferred are phenyl and naphthyl.

The substituent group which may be present on the aryl group includes,for example, halogen (e.g., fluorine, chlorine, etc.), linear orbranched C₁-C₅ alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl,tert-pentyl and the like), trifluoromethyl, linear or branched C₁-C₅alkoxy (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy,sec-butoxy, tert-butoxy, pentoxy, isopentoxy, neopentoxy, tert-pentoxyand the like), hydroxy, C₂-C₅ acyloxy (e.g., acetoxy, propionyloxy,butyryloxy, isobutyryloxy, valeryloxy and the like), carboxyl, and C₂-C₅acyl (e.g., acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl,pivaroyl and the like). Preferred are halogen and C₁-C₅ alkyl. The morepreferred are fluorine, chlorine, and methyl.

Preferred examples of the optionally substituted C₆-C₁₀ aryl group forR¹ are 4-fluorophenyl, 4-chlorophenyl, p-tolyl, and 2-naphthyl.

The linear or branched C₁-C₄ alkyl group mentioned for R² and R³includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl and the like. Preferred are propyl, isopropyl, andtert-butyl. The more preferred is isopropyl.

Referring to R² and R³, one of them is preferably hydrogen and the otheris propyl, isopropyl, isobutyl, or tert-butyl. More preferably, R² ispropyl, isopropyl, isobutyl, or tert-butyl and R³ is hydrogen. Stillmore preferably, R² is isopropyl and R³ is hydrogen.

The C₃-C₇ ring which may be formed jointly by R² and R³ includescyclopropylidene, cyclobutylidene, cyclopentylidene, cyclohexylidene,cycloheptylidene, and so forth. Cyclopentylidene and cyclohexylidene areparticularly preferred.

The lower alkyl group mentioned for R⁴ includes linear or branchedgroups having 1 to 6 carbon atoms, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl,neopentyl, tert-pentyl, hexyl, 4-methylpentyl, 1,1-dimethylbutyl,2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl and the like.Preferred are methyl and isobutyl. The above-mentioned lower alkyl groupmay be substituted by the following aryl group, cycloalkyl group, oraromatic heterocyclic residue.

The aryl group includes phenyl, 1-naphthyl, 2-naphthyl, and so forth.Particularly preferred is phenyl.

The cycloalkyl group preferably includes C₃-C₆ cycloalkyl; for example,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and so forth.Particularly preferred is cyclohexyl.

The aromatic heterocyclic residue includes heteromonocyclic residueseach containing at least one hetero atom selected from the groupconsisting of oxygen, nitrogen, and sulfur as a ring member andcorresponding fused heterocyclic residues. The heteromonocyclic residueincludes, but is not limited to, pyrrolyl, furyl, thienyl, oxazolyl,thiazolyl, imidazolyl, pyrazolyl, pyridyl and the like. The fusedheterocyclic residue includes, but is not limited to, indolyl, quinolyl,benzothienyl, benzofuryl, indazolyl, quinazolinyl, phthalazinyl,quinoxalinyl and the like. Particularly preferred is indolyl.

Preferred examples of the lower alkyl group which may be substituted byaryl, cycloalkyl or aromatic heterocyclic residue as expressed by R⁴ areisobutyl, benzyl, cyclohexylmethyl, and indol-3-ylmethyl.

Representative compounds of the formula (I) are

N-(2-naphthalenesulfonyl)-L-valyl-L-leucinal,

N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal,

N-(4-chlorophenylsulfonyl)-L-valyl-L-leucinal,

N-(4-methylphenylsulfonyl)-L-valyl-L-leucinal,

N-(4-fluorophenylsulfonyl)-L-valyl-L-phenylalaninal,

N-(2-naphthalenesulfonyl)-L-valyl-L-phenylalaninal,

N-(4-chlorophenylsulfonyl)-L-valyl-L-phenylalaninal,

N-(4-methylphenylsulfonyl)-L-valyl-L-phenylalaninal,

N-(4-chlorophenylsulfonyl)-L-valyl-L-tryptophanal,

N-(4-fluorophenylsulfonyl)-L-valyl-L-cyclohexylalaninal,

N-(2-naphthalenesulfonyl)-L-valyl-L-cyclohexylalaninal,

N-(4-chlorophenylsulfonyl)-L-valyl-L-cyclohexylalaninal, andpharmaceutically acceptable salts thereof.

Particularly preferred are

N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal, and a pharmacuticallyacceptable salt thereof.

The pharmaceutically acceptable salt of the compound of formula (I)includes salts with inorganic bases; for example, salts with alkalimetals such as sodium, potassium, etc., salts with alkaline earth metalssuch as calcium, magnesium, etc., aluminum salt, ammonium salt, etc.;salts with organic bases such as trimethylamine, pyridine, picoline,ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine,N,N-dibenzylethylenediamine, etc.; salts with inorganic acids such ashydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid,phosphoric acid, etc.; salts with organic acids such as formic acid,acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaricacid, maleic acid, citric acid, succinic acid, malic acid,methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,etc.; and salts with amino acids such as arginine, lysine, ornithine,aspartic acid, glutamic acid, and so forth.

The prophylactic and therapeutic drug of the present invention can beoptionally provided in any dosage form known in the art, that can bemanufactured by a known pharmaceutical technology which comprises, forexample, mixing or dissolving the active compound with apharmaceutically acceptable carrier or vehicle. Of such dosage forms,the oral dosage form for use in humans include powders, granules,tablets, capsules, syrups, and other liquid preparations. Powders,granules, tablets and the like can be manufactured using optionalpharmaceutically suitable carriers that are suitable for solidpreparations, such as excipients (e.g., starch, glucose, fructose,sucrose, lactose, etc.), lubricants (e.g., magnesium stearate, calciumstearate, etc.), disintegrators (e.g., starch, crystalline cellulose,etc.), binders (e.g., starch, gum arabic, etc.), and so forth. Suchsolid preparation may be optionally coated with a coating agent (e.g.,gelatin, sucrose, etc.) or an enteric coating (e.g.,hydroxypropylmethylcellulose phthalate, methacrylic copolymers, shellac,etc.), so that the active compound may be released specifically in thebowels. For the manufacture of syrups and other liquids, variousadditives such as stabilizers (e.g., sodium edetate etc.), suspendingagents (e.g., gum arabic, carmellose, etc.), corrigents (e.g., simplesyrup, glucose, etc.), perfumes, etc. can be appropriately added. Thedosage form for non-oral systemic administration includes injections,suppositories, etc. Injections can be manufactured by using solvents(e.g. water for injection, etc.), stabilizers (e.g., sodium edetateetc.), isotonizing agents (e.g., sodium chloride, glycerin, mannitol,etc.), pH control agents (e.g., hydrochloric acid, citric acid, sodiumhydroxide, etc.), suspending agents (e.g., methylcellulose, sodiumcarboxymethylcellulose, etc.), and other suitable additives. For themanufacture of suppository, a suppository base (e.g., cacao butter,macrogol, etc.) and the like may be appropriately used. The dosage formfor topical administration includes, for example, eye-drops andophthalmic ointments. For the manufacture of eye-drops and ophthalmicointments, a variety of known substances such as solvents (e.g.,physiological saline, sterile purified water, etc.), stabilizers (e.g.,sodium edetate, citric acid, etc.), emulsifiers (e.g.,polyvinylpyrrolidone), suspending agents (e.g.,hydroxypropylmethylcellulose, methylcellulose, hydroxymethylcellulose,etc.), surfactants (e.g., Polysorbate 80, hydrogenatedpolyethoxyethylene castor oil, etc.), preservatives (e.g., benzalkoniumchloride, p-hydroxybenzoic esters, chlorobutanol, etc.), buffers (e.g.,boric acid, borax (sodium borate), sodium acetate, citrate buffer,phosphate buffer, etc.), isotonizing agents (e.g., sodium chloride,glycerin, mannitol, etc.), pH control agents (e.g., hydrochloric acid,sodium hydroxide, etc.), and ointment bases (e.g., white petrolatum,lanolin, etc.) can be appropriately selected for use.

The prophylactic and therapeutic drug of the present invention is usefulfor the prevention and treatment of retinochoroidal diseases, glaucomaand ocular hypertension arising from ocular fundus tissue cytopathy, andposterior complications arising from photocoagulation. Theretinochoroidal diseases include, but are not limited to, retinalvascular abnormalities such as retinal vessel occlusion, retinalperiphlebitis, Eales' disease, ischemic eye syndrome, retinal arteriolarmacroaneurysm, etc.; retinopathy associated with hypertension or renaldisease; diabetic retinopathy; the retinal pigment epithelitis; retinaldystrophy; macular dystrophy; retinochoroidal atrophy;retinochoroiditis; macular degeneration; macular edema; retinal pigmentepithelial detachment; retinal detachment; degenerative retinoschisis;tumors such as retinoblastoma, tumors of the retinal pigment epithelium,capillary hemangioma of the optic nerve head, etc.; optic neuropathiessuch as ischemic optic neuropathy etc.; and optic disc swelling such aschoked disc/papilledema etc. The prophylactic and therapeutic drug ofthe present invention is particularly useful for the prevention andtreatment of retinal vessel occlusion and macular degeneration. Theglaucoma and ocular hypertension arising from ocular fundus tissuecytopathy include open angle glaucoma, low tension glaucoma, angleclosure glaucoma, etc. and ocular hypertension with visual fielddefects. The prophylactic and therapeutic drug of the present inventionis particularly useful for the prevention and treatment of low tensionglaucoma. The posterior complications arising from photocoagulationinclude macular edema, retinal detachment, optic neuritis, abnormalvisual field, abnormal light sense, or color vision defect.

The dosage of the compound of formula (I) or a pharmaceuticallyacceptable salt thereof according to the present invention is dependenton the target disease, clinical state and other conditions of patients,administration route, and other factors. Generally speaking, theobjective effect can be achieved in a general dose of 1-1000 mg,preferably 10-500 mg, for oral administration, or generally 0.1-300 mg,preferably 1-150 mg, for parenteral administration. For topicaladministration, an eye-drop having a concentration of 0.001-1.0 w/v %,preferably 0.01-0.5 w/v %, can be instilled in the eye by 20-50 μl perdose, with a frequency of about 5-6 doses a day.

As long as the object of the present invention is not impaired, theprophylactic and therapeutic drug of the present invention can be usedin combination with other active ingredients. As such other activeingredients, there can be mentioned anticoagulants such as warfarinpotassium, urokinase, aspirin, etc.; vasoprotectants/hemostatics such ascarbazochrome sodium sulfonate, etc.; peripheral vasodilators such askallidinogenase, pentoxifylline, sarpogrelate hydrochloride, tocopherolnicotinate, etc., adrenocorticoids such as betamethasone, dexamethasone,prednizolone, etc., antiphologistic enzyme preparations such asserrapeptase, streptokinase, streptodornase, etc., antiglaucoma drugssuch as β-blockers, mannitol, acetazolamide, etc., andantiprostaglandins.

EXAMPLE

The following examples and test examples are intended to illustrate thepresent invention in further detail and should by no means be construedas defining the scope of the invention. In the following examples andtest examples, Compound 1 means

N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal.

Example 1

Tablets Compound 1 50 mg Lactose 80 mg Starch 17 mg Magnesium stearate 3 mg Crystalline cellulose 10 mg

Tablets were prepared by a conventional method, which contained theabove ingredients as materials for one tablet. The tablets may beapplied with a conventional enteric coating (e.g.,hydroxypropylmethylcellulose phthalate), a sugar coating, or a film(e.g., ethylcellulose), as necessary.

Example 2

Capsules Compound 1 75 mg Mannitol 75 mg Starch 17 mg Calcium stearate 3 mg

The above ingredients as materials for one capsule were homogeneouslymixed, granulated by a conventional method, and filled into hardcapsules. The granules may be applied with a conventional entericcoating (e.g., hydroxypropylmethylcellulose phthalate), a sugar coating,or a film (e.g., ethylcellulose), as necessary prior to filling.

Example 3

Parenteral suspension Compound 1 750 mg Sodium carboxymethylcellulose500 mg Water for injection to make 100 ml

The above ingredients were aseptically admixed by a conventional methodto provide a parenteral suspension.

Example 4

Eye-drops Compound 1 50 mg Boric acid 700 mg Borax q.s. Sodium chloride500 mg Hydroxymethylcellulose 0.5 g Sodium edetate 0.05 mg Benzalkoniumchloride 0.005 mg Sterile purified water to make 100 ml

Test Example 1

Effect on Rat Ocular Fundus Tissue Cytopathy Method:

Using male SD rats (b. wt. 150 g), the central retinal artery wasoccluded with an aneurysm clip under anesthesia with 5 w/v % ketamineHCl/2 w/v % xylazine HCl (3:1, 0.5 ml, i.p.) to arrest the blood flowfor 75 minutes. In the normal group, the central retinal artery wasexposed but ischemia was not introduced. Tissue specimens were prepared5 days after reperfusion. For use as a tissue specimen, the enucleatedeyeball was fixed in 4% formaldehyde and embedded in paraffin in aroutine manner. Then, thin sections (3 μm thick) were prepared bytransverse slicing at right angles with the retinal surface includingthe optic disc and stained with hematoxylin-eosin. Under the lightmicroscope, the retinal neurons (ganglion cells) and inner nuclear layercells were counted per 0.25 mm span of the retinal section at apredetermined distance (1-2 mm) from the optic disc. As the test drug, aparenteral suspention prepared as in Example 3 was administeredintraperitoneally in a dose of 100 mg, as Compound 1, per kg body weight15 minutes before arterial occlusion, immediately after reperfusion, andthereafter once daily (Compound 1 administration group). The control andnormal groups were similarly given the vehicle used in Example 3. In thepositive control group, Cbz-Val-Phe-H [S.C. Hong et al., Stroke, 25 (3),663-669, 1994], 100 mg/kg, was similarly administered (MDLadministration group).

Results:

The results are shown in FIGS. 1 and 2.

As compared with the normal group, both the ganglion cell count andinner nuclear layer cell count were significantly decreased by arterialocclusion. An administration of Compound 1 significantly inhibited thedereases in ganglion and inner nuclear layer cell counts caused by thearterial occlusion. In the MDL administration group, the degree ofinhibition was slight.

The above results indicate that the active Compound 1 of the presentinvention is effective in improving ocular fundus tissue cytopathy.

Test Example 2

Effect on Rat Enucleated Retina Cytopathy Under Hypoxia Method:

The ocular fundus tissue (retina) of male SD rats (b. wt. 150 g) wasremoved and incubated under hypoxia in RPMI 1640 medium (manufactured by(Gibco) without glucose. The ocular fundus tissue was homogenized 3 and6 hours later, and the cleavage product of endocellular spectrin wasisolated by SDS electrophoresis and detected using homophil prepared bythe method of Saido et. al. [The Journal of Biological Chemistry, 268,25239-25243 (1993)]. As the test compounds, Compound 1 and MDL weredissolved in ethanol to a concentration of 20 mM and added to theculture medium to a concentration of 100 μM (Compound 1 administrationgroup and MDL administration group). As a control, the ocular fundustissue of male SD rats (b. wt. 150 g) was incubated in the same mannerin the presence of oxygen in RPMI 1640 medium containing glucose.

Results:

The results are shown in FIGS. 3(a) and 3(b).

When the tissue was incubated in a glucose-containing culture medium inthe presence of oxygen, cleavage of spectrin was not observed. Incontrast, when the tissue was incubated for 3 hours and 6 hours in aglucose-free culture medium under hypoxia, spectrin shown by a 220 kDaband was cleaved by calpain, which resulted in the detection of thecleavage products of spectrin as shown by 145 and 150 kDa bands. Whenthe tissue was incubated in a culture medium supplemented with Compound1 and MDL, which are calpain inhibitors, although a 150 kDa band due tothe digestion of spectrin was somewhat detected, a 145 kDa band was notdetected.

The foregoing results indicate elevation of calcium concentrations inthe ocular fundus tissue due to the absence of oxygen, which leads tothe activation of calpain, that is a calcium-dependent protease, andinjury of the cells. The results clearly shows amelioration of theinjury by Compound 1.

INDUSTRIAL APPLICABILITY

The prophylactic and therapeutic drug of the present invention is usefulfor the prevention and treatment of diseases arising from ocular fundustissue cytopathy, e.g., retinal vascular abnormalities such as retinalvessel occlusion, retinal periphlebitis, Eales' disease, ischemic eyesyndrome, retinal arteriolar macroaneurysm, etc.; retinopathy associatedwith hypertension or renal disease; diabetic retinopathy; retinalpigment epithelitis; retinal dystrophy; macular dystrophy;retinochoroidal atrophy; retinochoroiditis; macular degeneration;macular edema; retinal pigment epithelial detachment; retinaldetachment; degenerative retinoschisis; tumors such as retinoblastoma,tumors of the retinal pigment epithelium, capillary hemangioma of theoptic nerve head, etc.; optic neuropathies such as ischemic opticneuropathy etc.; optic disc swelling such as choked disc/papilledemaetc.; glaucoma such as open angle glaucoma, low tension glaucoma, angleclosure glaucoma; and ocular hypertension with visual field defects,posterior complications arising from photocoagulation such as macularedema, retinal detachment, optic neuritis, abnormal visual field,abnormal light sense, and color vision defect.

The present invention is based on Application No. 53624/1998 filed inJapan, the content of which is incorporated hereinto by reference.

What is claimed is:
 1. A method for the prophylaxis or treatment of adisease arising from ocular fundus tissue cytopathy, which comprisesadministering an effective amount of a compound of the formula (I)

wherein R¹ represents an alkyl group having 1 to 4 carbon atoms or anaryl group having 6 to 10 carbon atoms which is optionally substituted;R² and R³ may be the same or different and each represents hydrogen oran alkyl group having 1 to 4 carbon atoms or R² and R³ may jointly forma ring having 3 to 7 carbon atoms; and R⁴ represents a lower alkyl groupwhich is optionally substituted by aryl, cycloalkyl, or aromaticheterocyclic residue, or a pharmaceutically acceptable salt thereof. 2.The method as claimed in claim 1, wherein R¹ in the formula (I) isphenyl or naphthyl, which may be substituted by fluorine, chlorine, ormethyl.
 3. The method as claimed in claim 1, wherein R¹ in the formula(I) is a group selected from the group consisting of methyl,4-fluorophenyl, 4-chlorophenyl, p-tolyl, and 2-naphthyl.
 4. The methodas claimed in claim 1, wherein, in the formula (I), R² is propyl,isopropyl, or tert-butyl and R³ is hydrogen.
 5. The method as claimed inclaim 1, wherein, in the formula (I), R² is isopropyl and R³ ishydrogen.
 6. The method as claimed in claim 1, wherein, in the formula(I), R⁴ and R³ jointly form cyclopentylidene or cyclohexylidene.
 7. Themethod as claimed in claim 1, wherein, in the formula (I), R⁴ is a groupselected from the group consisting of isobutyl, benzyl,cyclohexylmethyl, and indol-3-ylmethyl.
 8. The method as claimed inclaim 1, wherein the compound isN-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal or a pharmaceuticallyacceptable salt thereof.
 9. The method as claimed in any of claim 1,wherein the disease arising from ocular fundus tissue cytopathy is adisease selected from the group consisting of retinochoroidal disease,glaucoma, and posterior complications arising from photocoagulation. 10.The method as claimed in to claim 9, wherein the disease arising fromocular fundus tissue cytopathy is retinochoroidal disease.
 11. Themethod as claimed in claim 10, wherein the retinochoroidal disease is amember selected from the group consisting of retinal vessel occlusion,retinal periphlebitis, Eales' disease, ischemic eye syndrome, retinalarteriolar macroaneurysm, retinopathy associated with hypertension orrenal disease, diabetic retinopathy, retinal pigment epithelitis,retinal dystrophy, macular dystrophy, retinochoroidal atrophy,retinochoroiditis, macular degeneration, macular edema, retinal pigmentepithelial detachment, retinal detachment, degenerative retinoschisis,retinoblastoma, tumors of the retinal pigment epithelium, capillaryhemangioma of the optic nerve head, ischemic optic neuropathy, andchoked disc/papilledema.
 12. The method as claimed in claim 11 whereinthe retinochoroidal disease is retinal vessel occlusion.
 13. The methodas claimed in claim 11, wherein the retinochoroidal disease is maculardegeneration.
 14. The method as claimed in to claim 9 wherein thedisease arising from ocular fundus tissue cytopathy is a posteriorcomplication arising from photocoagulation.
 15. The method as claimed inclaim 14, wherein the posterior complication arising fromphotocoagulation is a member selected from the group consisting ofmacular edema, retinal detachment, optic neuritis, abnomal visual field,abnormal light sense, and color vision defect.
 16. The method as claimedin to claim 9, wherein the disease arising from ocular fundus tissuecytopathy is glaucoma.
 17. The method as claimed in claim 16, whereinthe glaucoma is a member selected from the group consisting of openangle glaucoma, low tension glaucoma, and angle closure glaucoma. 18.The method as claimed in claim 17, wherein the glaucoma is low tensionglaucoma.